HIV-1 encodes genes that are crucial for replication in primary cells exerting functions not provided by the host. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is now strong evidence that these proteins operate in conjunction with specific host factors. In fact, none of the HIV accessory proteins has a known enzymatic activity. Instead, these proteins function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. Over the past few years, our research focus has shifted more and more towards the characterization of host factors and their roles in virus replication. One of the factors we recently identified is human mannose receptor I (hMRC1), a protein expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages and is involved in the endocytic uptake of HIV-1 into these cells. In FY19 we continued a project characterizing the function of hMRC1 in the control of HIV-1 replication in macrophages. We previously identified hMRC1 as a novel macrophage-specific restriction factor that inhibits virus release through a BST-2-like mechanism. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1-imposed restriction suggesting the involvement of additional cellular factor(s) in the process. These data were published in Cell Reports (Sukegawa et al., 2018). Efforts are still ongoing to identify and characterize the additional cellular factors involved in hMRC1-mediated restriction of virus release using proteomics approaches. In addition, we continued work on a second effect of hMRC1 that influences the infectivity of viruses produced in the presence of hMRC1. Unlike the effect on virus release, this effect of hMRC1 is virus-specific and affects primarily R5-tropic virus isolates. We have initiated studies to determine the possible involvement of chemokine receptors in this process. Finally, we continue our effort to fully understand the mechanism of HIV-mediated transcriptional silencing of hMRC1. We have systematically ruled out the contribution of most HIV-encoded accessory proteins, including Vpr, and are now focusing on other viral components, in particular Tat. Aside from our work on hMRC1, we continued our characterization of proteins we identified in the process of a proteomic analysis of Vpr-interacting host factors. We have studied the effects of transient over-expression of these factors on HIV replication. We are now in the process of assessing the effects of silencing these genes. Since Vpr effects are typically not seen in immortalized cell lines, we have put extra effort into establishing CRISPR/Cas9-based knockout technology to allow measuring effects of our candidate genes during spreading infection of HIV-1 in primary cells. We had some success with silencing cellular genes in monocyte-derived macrophages and we are currently optimizing our technique for maximal effect. Aside from our own experiments, we have been involved in collaborative efforts with several international partners. In one published study, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We found that that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition (ref). In a second collaboration, we studied the evolution of Vpu with regard to its effect on BST-2/tetherin. Some SIVs such as SIVcpz, SIVgsn, SIVrcm, or SIVmus encode a vpu gene. We found that the Vpu proteins from most of these SIVs fail to antagonize BST-2. Only Vpu from SIVgsn was able to antagonize human BST-2 as well as the BST-2 from its natural host. The results from this study provide interesting novel insights into the evolution of the anti-BST-2 activity of Vpu. The study has been conditionally accepted for publication. Finally, in collaboration with the Clouse lab, we studied the effects of cytokines on filovirus Env-mediated infection of human macrophages. We found that pre-incubation of primary human monocyte-derived macrophages with interleukin-10 significantly enhanced filovirus entry in cells obtained from multiple healthy donors. Results from this collaboration also have been conditionally accepted for publication.